Single-stranded Bubble Research Articles

Architecture of the Yeast RNA Polymerase II Open Complex and Regulation of Activity by TFIIF

To investigate the function and architecture of the open complex state of RNA polymerase II (Pol II), Saccharomyces cerevisiae minimal open complexes were assembled by using a series of heteroduplex HIS4 promoters, TATA binding protein (TBP), TFIIB, and Pol II. The yeast system demonstrates great flexibility in the position of active open complexes, spanning 30 to 80 bp downstream from TATA, consistent with the transcription start site scanning behavior of yeast Pol II. TFIIF unexpectedly modulates the activity of the open complexes, either repressing or stimulating initiation. The response to TFIIF was dependent on the sequence of the template strand within the single-stranded bubble. Mutations in the TFIIB reader and linker region, which were inactive on duplex DNA, were suppressed by the heteroduplex templates, showing that a major function of the TFIIB reader and linker is in the initiation or stabilization of single-stranded DNA. Probing of the architecture of the minimal open complexes with TFIIB-FeBABE [TFIIB-p-bromoacetamidobenzyl-EDTA-iron(III)] derivatives showed that the TFIIB core domain is surprisingly positioned away from Pol II, and the addition of TFIIF repositions the TFIIB core domain to the Pol II wall domain. Together, our results show an unexpected architecture of minimal open complexes and the regulation of activity by TFIIF and the TFIIB core domain.

Flanking sequence effects on oligonucleotide hybridization

Microarray platforms measure both sequence content and prevalence of sequence by leveraging oligonucleotide hybridization. Computational probe design centers on short regions of high sequence similarity, most often between 25 and 50 base pairs in length. A variety of pattern-matching algorithms specific to the probe length are employed to determine the uniqueness of a target against its background, and a subset of these consider internal structure of the probe as a contra-indicator of successful hybridization. Since it is assumed that the target structure will mirror that of the probe, no consideration is given to the flanking sequences. In this study, we start to examine on the effects of total target sequence length in the context of common hybridization assay conditions. There are two considerations: due to the flexible nature of the single-stranded backbone, loops and bubbles can still yield stable hybridization between species with less than perfect sequence homology; the flanking sequences may loop back and occupy or obstruct the intended duplex region. Selecting a small subset of 33mer probes from the Affymetrix SNP6.0 Array we generated a pool of potentially cross-hybridizing sequence regions found by using the SeqNFindTM platform. Pools of length variant targets centered around the potentially cross hybridizing sequence were generated from human reference genome 36.3 and computationally examined using OMPTM to calculate the affinity constants. To demonstrate that the modeled properties affect measurements a small subset of these targets and probes were tested on microarrays.

Reverse Gyrase Functions as a DNA Renaturase: ANNEALING OF COMPLEMENTARY SINGLE-STRANDED CIRCLES AND POSITIVE SUPERCOILING OF A BUBBLE SUBSTRATE

Reverse gyrase is a hyperthermophile-specific enzyme that can positively supercoil DNA concomitant with ATP hydrolysis. However, the DNA supercoiling activity is inefficient and requires an excess amount of enzyme relative to DNA. We report here several activities that reverse gyrase can efficiently mediate with a substoichiometric amount of enzyme. In the presence of a nucleotide cofactor, reverse gyrase can readily relax negative supercoils, but not the positive ones, from a plasmid DNA substrate. Reverse gyrase can completely relax positively supercoiled DNA, provided that the DNA substrate contains a single-stranded bubble. Reverse gyrase efficiently anneals complementary single-stranded circles. A substoichiometric amount of reverse gyrase can insert positive supercoils into DNA with a single-stranded bubble, in contrast to plasmid DNA substrate. We have designed a novel method based on phage-mid DNA vectors to prepare a circular DNA substrate containing a single-stranded bubble with defined length and sequence. With these bubble DNA substrates, we demonstrated that efficient positive supercoiling by reverse gyrase requires a bubble size larger than 20 nucleotides. The activities of annealing single-stranded DNA circles and positive supercoiling of bubble substrate demonstrate that reverse gyrase can function as a DNA renaturase. These biochemical activities also suggest that reverse gyrase can have an important biological function in sensing and eliminating unpaired regions in the genome of a hyperthermophilic organism.

Single-Strand-Specific Radiosensitization of DNA by Bromodeoxyuridine

The effects of bromodeoxyuridine (BrdUrd) substitution for thymidine on gamma-ray-induced strand breakage were determined in single- and double-stranded oligonucleotides and double-stranded oligonucleotides containing a mismatched bubble region. BrdUrd does not sensitize complementary double-stranded DNA to gamma-ray-induced strand breakage, but it greatly sensitizes single-stranded DNA. However, when the BrdUrd is present in a single-stranded bubble of a double-stranded oligonucleotide, the non-base-paired nucleotides adjacent to the BrdUrd as well as several unpaired sites on the opposite unsubstituted strand are strongly sensitized. The radiosensitization properties of BrdUrd result primarily from the electrophilic nature of the bromine, making it a good leaving group and leading to the irreversible formation of the uridine-yl radical (dUrd(.)) or the uridine-yl anion (dUrd(-)) upon addition of an electron. The radiolytic loss of the bromine atom is greatly suppressed in double-stranded compared to single-stranded DNA. Thus we propose that the radiosensitization effects of bromouracil in vivo will likely be limited to single-strand regions such as found in transcription bubbles, replication forks, DNA bulges and the loop region of telomeres. Our results may have profound implications for the clinical use of bromodeoxyuridine (BrdUrd) as a radiosensitizer as well as for the development of targeted radiosensitizers.

Topology matters: Some aspects of DNA physics

ABSTRACTBiological cells, in some sense, are all about topology: Biomembranes separating different volumes from one another, ions or even macromolecules having to cross these membranes in controlled fashion through membrane pores; or certain proteins moving along the DNA to find their target sequence instead of searching for this site in the full 3-dimensional cell volume. Even on the single biopolymer level, topology is an essential ingredient: Intriguingly, in bacteria DNA occurs knotted, i.e., in a state topologically different from a simply connected ring. It is a key question to understand the statistical behaviour of such knotted DNA to understand a number of physiological processes having to overcome this knottedness, or to quantify results from DNA separation techniques such as electrophoresis, in which the knottedness influences the mobility. At the same time, double-stranded DNA continuously opens up floppy single-stranded bubbles, which fluctuate in size, exposing the single Watson-Crick bases to binding proteins. Again, statistical mechanical tools can be employed to examine the bubble dynamics. Here, we introduce some recent results on DNA knots and bubble fluctuations.

Construction of a gene library with mung bean nuclease-treated genomic DNA.

AbstractMung bean nuclease (MBN) has been used typically for its single-stranded nuclease activity (1). Under defined reaction conditions, however, which include altered solvation and elevated temperature, hypersensitive sites surrounding coding regions become sensitive to nuclease cleavage (2), while sequences within coding regions remain insensitive Fig. 1). Mung bean cleavage of genomic DNA from Plasmodium species results in a solution of gene-containing fragments Fig. 2) (2). A significant proportion of the noncoding regions is reduced to shards, but it is not known what percentage of the genome this includes. The cleavage sites are not simply single-stranded bubbles in the DNA but stable nucleic acid structures that contain specific arrangements of paired and unpaired nucleotides (3). Size of mung bean vs protein coding regions. Data points from mung bean fragments are shown as dotted circles. Data points from published accounts of other laboratories are shown as open circles. The solid line represents the linear equation, y = 1.2x - 338 derived from the data. The shaded line represents y = x, the expected result if the mung bean fragment sizes were the same size as the coding region. Mung bean nuclease treated of DNA yields products that run as unit bands upon agarose electrophoresis. A plasmid, pPbSL7.8 (2) was treated with EcoRI (lane a), mung bean nuclease and EcoRI (lane b) or mung bean nuclease alone (lane c). The plasmid contains a DNA fragment from P. berghei. The fragment is 7.8 kb and contains the external transcribed spacer, the small subunit rRNA, ITS1, 5.8S RNA, ITS2, and 3 kb of the LSU rRNA. KeywordsMung BeanPlasmodium SpeciesCesium ChlorideMung Bean NucleaseSaponin SolutionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A hybrid bacterial replication origin.

We constructed a hybrid replication origin that consists of the main part of oriC from Escherichia coli, the DnaA box region and the AT-rich region from Bacillus subtilis oriC. The AT-rich region could be unwound by E. coli DnaA protein, and the DnaB helicase was loaded into the single-stranded bubble. The results show that species specificity, i.e. which DnaA protein can do the unwinding, resides within the DnaA box region of oriC.

GreA-induced transcript cleavage is accompanied by reverse translocation to a different transcription complex conformation.

GreA- and GreB-induced transcript cleavage drives reverse translocation of Escherichia coli RNA polymerase on a DNA template in the absence of NTPs (Feng, G.-H., Lee, D. N., Wang, D., Chan, C. L., and Landick, R. (1994) J. Biol. Chem. 269, 22282-22294, accompanying report). During transcript elongation, the sizes of the DNA footprint and the single-stranded transcription bubble vary markedly among transcription complexes halted at different template positions. To test whether transcription complex intermediates formed during transcript cleavage-induced reverse translocation also display heterogeneous conformations at different template positions, we examined the structures of two different transcription complexes before and after GreA treatment. Transcription complexes halted at position +16 after initiation at the T7 A1 promoter or paused at the trpL pause site exhibited strong blocks to transcript cleavage after removal of 6 to 10 nucleotides. In both cases, the down-stream contact between RNA polymerase and DNA moved little during transcript cleavage, thereby increasing its distance from the active site, whereas the upstream DNA contact and the borders of the transcription bubble moved in approximate register with the transcript 3'-end. The backward movements of halted E. coli RNA polymerase are similar to a recently postulated model for discontinuous translocation during transcription, but differ from those reported for arrested RNA polymerase II transcription complexes.

Easily unwound DNA sequences and hairpin structures in the Epstein-Barr virus origin of plasmid replication.

The Epstein-Barr virus (EBV) origin of plasmid replication (oriP) includes two known cis-acting components, the dyad symmetry region and the family of repeats. We used P1 nuclease, a single-strand-specific endonuclease, to probe EBV oriP for DNA sequences that are intrinsically easy to unwind on a negatively supercoiled plasmid. Selective nuclease hypersensitivity was detected in the family of repeats on an oriP-containing plasmid and in the dyad symmetry region on a plasmid that lacks the family of repeats, indicating that the DNA in both cis-acting components is intrinsically easy to unwind. The hierarchy of nuclease hypersensitivity indicates that the family of repeats is more easily unwound than the dyad symmetry region, consistent with the hierarchy of helical stability predicted by computer analysis of the DNA sequence. A specific subset of the family of repeats is nuclease hypersensitive, and the DNA structure deduced from nucleotide-level analysis of the P1 nuclease nicks is a cruciform near a single-stranded bubble. The dyad symmetry region unwinds to form a broad single-stranded bubble containing hairpins in the 65-bp dyad sequence. We propose that the intrinsic ease of unwinding the dyad symmetry region, the actual origin of DNA replication, is an important component in the mechanism of initiation.

Instability of the nuclear chromatin of procyclic Trypanosoma brucei brucei

Digestion of nuclear chromatin of Trypanosoma brucei brucei procyclic culture forms with micrococcal nuclease yielded DNA fragments which formed DNA ladders in agarose gels, similar to those of rat liver. However, the chromatin of trypanosomes was digested more rapidly. The digestion of T. b. brucei chromatin yielded a large amount of DNA fragments of core-particle size. The numbers of base pairs per nucleosomal and linker DNA were identical in both species, if the digestion conditions were reduced in the case of T. b. brucei. Psoralen cross-linking of soluble chromatin of trypanosomes at 5 mM salt at pH 7 or pH 10 resulted in an irregular array of single-stranded (ss) bubbles separated by variable stretches of double-stranded (ds) DNA. The proportion of ss DNA was low compared with the ratio of ss/ds stretches in rat liver chromatin, which also showed regularly arranged nucleosomal DNA. Soluble chromatin of T. b. brucei, pre-treated with 500 mM NaCl to remove a potential H1 and psoralen cross-linked at 5 mM salt at pH 7 or pH 10 was to a great extent ds in both situations. The true nucleosome filament organization of T. b. brucei chromatin could only be shown by psoralen cross-linking the DNA in whole nuclei under physiological conditions. The results indicate that the chromatin of procyclic T. b. brucei differs significantly in its compaction pattern from rat liver chromatin; a typical histone H1 is not found, and the DNA-protein interactions are also less stable and can more easily be destabilized by experimental conditions.

Influence of DNA sequence and supercoiling on the process of cruciform formation

We have studied some of the effects of DNA sequence and negative superhelicity on the rate of cruciform formation. Replacing the sequence AATT at the center of a perfect 68 base-pair palindromic sequence with the sequence CCCGGG decreases the rate of cruciform formation by a factor of at least 100. The logarithm of the rate constant of cruciform formation was found to increase linearly with linking difference. For the 68 base-pair perfect palindrome in a 4400 base-pair plasmid, each additional negative superhelical turn increased the rate of cruciform formation by a factor of 1.6. These results are consistent with a mechanism in which cruciform formation is initiated by the formation of a single-stranded bubble, 10 base-pairs in length, near the center of the palindromic sequence. In addition, we have examined the effect of introducing an asymmetric insertion into the palindromic sequence.

Structure of in-vivo transcribing chromatin as studied in simian virus 40 minichromosomes

In order to study the structure of chromatin during transcription, individual in-vivo transcribing simian virus 40 (SV40) minichromosomes were analyzed in the electron microscope after crosslinking the nascent RNA strands with different psoralen derivatives to the template DNA. Since psoralen crosslinks the DNA between nucleosomes, spreading of the crosslinked DNA and DNA-RNA complexes reveals single-stranded bubbles at positions where nucleosomes were located. We found that the transcribing SV40 minichromosomes contained a similar number of nucleosomes as did the minichromosomes without crosslinked nascent RNA. The nascent RNA was crosslinked in about equal proportions either in single-stranded bubbles of nucleosomal length or in continuously crosslinked regions between bubbles, in contrast with control experiments with ribosomal chromatin of Dictyostelium. Treatment of SV40 minichromosomes with 1.2 M-NaCl before and during photocrosslinking with psoralen led to the disappearance of the single-stranded bubbles. Since no bubbles could be detected at the attachment sites of the RNA molecules when the nucleosomes were disrupted in high salt, and since in about half of the molecules the RNA was attached to fully crosslinked linker DNA, we assume that the single-stranded bubbles with crosslinked RNA are not due to protection by the elongating RNA polymerase II complex, but are rather due to nucleosome-like structures. At the resolution level of single nucleosomes, these results imply for the first time that nucleosome-like structures (perhaps modified compared with "normal" nucleosomes) on SV40 minichromosomes do not prevent transcription elongation by RNA polymerase II.

Psoralen-crosslinking of soluble and of H1-depleted soluble rat liver chromatin

We purified soluble rat liver chromatin and H1-depleted chromatin and photocrosslinked its DNA with psoralen at pH 7. Digestion of this chromatin with micrococcal nuclease produced a normal nucleosomal repeat. Chromatin was photoreacted in the presence of 0 to 700 mM-NaCl and was fractionated in sucrose gradients containing the same NaCl concentrations. The dissociation of H1 occurred as in the non-crosslinked controls and no preferential dissociation of core histones was observed. The samples between 100 and 500 mM-NaCl showed precipitation. In the electron microscope, the fibers appeared indistinguishable from the controls at low ionic strength. In the presence of 40 mM-NaCl, the fibers of the photoreacted chromatin were slightly more compact than the controls, and at 500 mM-NaCl, despite the complete dissociation of H1, there were still apparently intact fibers at this ionic strength. The disruption of the psoralen-treated chromatin fibers occurred only in 600 mM-NaCl, as opposed to 500 mM-NaCl in controls. The DNA of all the photoreacted samples was spread for electron microscopy under denaturing conditions. They revealed, for all the samples, single-stranded bubbles corresponding to 200 to 400 base-pairs in size. H1-depleted chromatin containing stoichiometric amounts of core histones was photoreacted at pH 10 and very low ionic strength. Under these conditions many of the nucleosomes appeared to be unraveled, although to a variable extent. In the electron microscope, the purified DNA from these samples showed extensive crosslinking when spread under denaturing conditions. These observations show that histone-DNA interactions different from those in intact nucleosomes may be created, which allow extensive access of psoralen to the DNA.

Psoralen-crosslinking of DNA as a probe for the structure of active nucleolar chromatin

Trimethylpsoralen was used to crosslink the extrachromosomal ribosomal DNA in nucleoli or nuclei of growing Dictyostelium discoideum cells. The DNA was extracted and was examined by spreading under denaturing conditions for electron microscopy. Intact 95,000 base ribosomal DNA molecules were seen, showing regularly spaced, single-stranded bubbles of about 200 to 400 bases in size, interrupted twice by 11,000 base heavily crosslinked stretches, which correspond to the known positions of the coding regions. The bubbles on the nontranscribed regions indicate the presence of nucleosomes during crosslinking. The DNA was digested with restriction enzymes and analysed by gel electrophoresis in parallel with DNA not treated with psoralen. Fragments from the non-coding region had the same mobility as untreated DNA, while those from the coding region had a markedly lower mobility, though not as low as that of crosslinked pure DNA. This shifting of the bands, specific to the coding region, was also seen when whole cells were treated with psoralen. Treatment of nucleoli with 2 m-NaCl (which is known to dissociate histones) before addition of psoralen led to strong crosslinking all along the ribosomal DNA, resulting in a decreased electrophoretic mobility of bands from the non-coding region, but no further retardation of those from the coding region. In differentiating Dictyostelium cells, slugs, where ribosomal RNA synthesis is very much reduced, the extent of psoralen-crosslinking in the coding region was reduced, but not completely to the level of that of the non-transcribed spacer. In order to test whether psoralen itself alters chromatin structure, crosslinked and non-crosslinked nucleoli from growing cells were lysed with heparin and spread for electron microscopy. There was no difference in the appearance or the frequency of the transcription units seen. Digestion of crosslinked nuclei with micrococcal nuclease indicated an undisturbed structure for bulk chromatin, as well as for the chromatin in the non-transcribed spacer of the ribosomal DNA. Thus psoralen-crosslinking does not lead to extensive disruption or distortion of the structure of either inactive or active chromatin. We conclude, taking the results presented in the Appendix into account, that the extent of psoralen-crosslinking in chromatin DNA is diagnostic for the structure of undistorted chromatin.(ABSTRACT TRUNCATED AT 400 WORDS)

The invertible G segment of phage Mu

Near the right or “variable” end of Mu DNA is a 3 kb segment, the G segment shown in the figure below, which inverts via intramolecular recombination between small regions of homology located at its ends (Daniel1 et al., Virology 57, 237-239, 1973; Hsu and Davidson, Virology 58, 229-239, 1974). Phage DNA containing the G segment in opposite orientations was originally detected by the presence of single-stranded bubbles near one end of Mu DNA heteroduplexes (Daniel1 et al., op. cit.). More recently a DNA restriction assay has been used to quantitate the proportion of phages with each orientation of the G segment; the assay is based on measurements of the relative quantities of two unique DNA restriction fragments which arise by cleavage of Mu DNA at a site outside the G segment and at a site located asymmetrically within the G segment, for example the Pst I and Kpn I sites shown in the figure below (Bukhari and Ambrosio, Nature 277, 575-577, 1978; Kamp et al., Nature 277, 577-580, 1978). When Daniell, Boram and Abelson (Proc. Nat. Acad. Sci. 70, 2153-2156, 1973) found that Mu DNA in phage grown by infection of E. coli K12 had the G segment primarily (99%) in one orientation, whereas DNA from phage grown by induction contained the G segment in both orientations (25-75% of each), they suggested that one orientation might be preferred for lytic growth by infection. This hypothesis was supported by the finding of Chow, Kahmann and Kamp (J. Mol. Biol. 7 73, 591-609, 1977) that viable plaqueforming Mu phages which were deleted for portions of the G and/or /3 segments, and were therefore unable to invert the G segment, all contained their G segments in the “lytic infection” orientation regardless of the method of growth; this orientation was defined as G(+). Their analysis of these deleted phages demonstrated that G segment inversion per se was not required for phage viability and that the rightmost 1.3 kb of the G segment could be deleted with no effect on phage growth in E. coli K12. One phage, with the 445-5 deletion shown in the figure below, contained an intact G segment but could not invert it; however, this phage could be complemented for G inversion by a nondeleted Mu prophage (Kamp et al., op. cit.). Using lysogens of MuA445-5 with the G segment frozen in either the G(+) or G(-) orientation, Kamp et al. (op. cit.) demonstrated that phage particles were released after induction of both lysogens but that only those containing G(+) DNA were able to make plaques on E. coli K12. Studies of complementation for inversion of the G segment in MuA445-5 revealed that a specific Mu function, gin (G inversion), was required for inversion and that this function was located in the p DNA segment adjacent to the G segment (Kamp et al., Cold Spring Harbor Symp. 43, 1159-l 167, 1979). Similar complementation studies revealed functional homology between several such invertible DNA systems. Kamp et al. (Cold Spring Harbor Symp. 43, 1159-l 167, 1979) found that phages Pl, P7 and

Modification of PM2 DNA with N-acetoxy-2-acetylaminofluorene: Changes in buoyant density, electrophoretic mobility and electron microscopic structure

PM2 DNA, forms I and II, were chemically modified with the ultimate carcinogen N-acetoxy-2-acetylaminofluorene. The products were subjected to 3 analytical procedures: (i) buoyant density centrifugation in the presence of the intercalating dyes ethidium bromide or propidium diiodide; (ii) agarose gel electrophoresis: and (iii) electron microscopy. It was found that carcinogen-modified DNAs banded at higher densities in the presence of either dye. This effect was far more pronounced with form I DNA than with form II. Form I DNA showed a concentration-dependent decrease of migration velocity in agarose gel electrophoresis after modification; form II DNA exhibited a similar but smaller effect. Electron microscopy of form I DNA did not reveal a consistent correlation between the extent of chemical modification and the loss of superhelical turns. Form II DNA exhibited characteristic alterations such as aggregation of several PM2 molecules into network-like structures, kinky configuration, and probably single-stranded bubbles extending over 600-1500 bases.